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Arora et al. Sustainable Chemical Processes (2014) 2:25 DOI 10.1186/s40508-014-0025-y

REVIEW Open Access promiscuity: using the dark side of enzyme specificity in white biotechnology Benu Arora1, Joyeeta Mukherjee1 and Munishwar Nath Gupta2*

Abstract Enzyme promiscuity can be classified into promiscuity, condition promiscuity and catalytic promiscuity. Enzyme promiscuity results in far larger ranges of organic compounds which can be obtained by . While early examples mostly involved use of , more recent literature shows that catalytic promiscuity occurs more widely and many other classes of can be used to obtain diverse kinds of molecules. This is of immense relevance in the context of white biotechnology as enzyme catalysed reactions use greener conditions. Keywords: Enzyme specificity, Catalytic promiscuity, Enzymes in organic synthesis, Enantioselectivity, Green chemistry

is concerned. The most dramatic departure from the “I suspect they put Socrates to death because there is classical concept of enzyme specificity is seen in the something terribly unattractive, alienating and non- phenomenon of catalytic promiscuity. This refers to human in thinking with too much clarity.”- the same enzyme catalyzing very different kinds of bio- transformations [3-6]. The Bed of Procrustes by Nassim Nicholas Taleb

Classification of enzymes and enzyme specificity Introduction Most of the people have forgotten (understandable since it The basic tenet of white biotechnology is to minimize disappeared from the standard texts many decades back!) damage to the environment rather than taking recourse that initially proteins were classified according to their to remediation as an “end of the pipe” solution [1,2]. En- solubility in various solvents. The five classes of proteins zymes either in isolated form or in the form of whole cells were albumins (soluble in water and salt solutions), globu- can play an important role because of their two well lins (sparingly soluble in water but soluble in salt solu- known virtues. The biocatalysts normally do not require tions), prolamines (soluble in 70-80% ethanol), glutenins high temperature or other conditions which involve high (soluble in acid or alkali) and scleroproteins (insoluble in consumption of energy. The biocatalysts are believed to aqueous solvents) [7]. As our knowledge of proteins grew, be fairly specific, which would mean less number of side the distinction between these various classes became fluid reactions. Side reactions lead to side products which lower and in fact confusing. The enzymes, in the early phase the atom economy of the reactions. These side reactions of biochemistry became the most important and most hence lower the yield of the desired product (making the studied class of proteins. These were named in a random catalysis less efficient) and necessitate complicated down- fashion just as people name buildings, streets and monu- stream processing. ments. Many of these names persist e.g. , , This review discusses the paradigm shifts over the years etc. Many of these names were based upon the in our understanding of the enzyme specificity. It also ex- nature of their catalytic activity. Lysozyme is named so as plains why so called lack of specificity is also a good news it lyses cells. So, the nomenclature and catalytic specificity as far as the usefulness of enzymes in biotechnology have a long history in the area of biocatalysis. The idea of a particular structure being absolutely related * Correspondence: [email protected] to a biological activity has been considered the hallmark of 2Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India biology for many decades. Hence, it is not surprising that Full list of author information is available at the end of the article enzyme commission classification was based upon the type

© 2014 Arora et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 2 of 9

of reaction the enzyme catalyzes –hydrolysis, isomerisation “alternative substrates”, hydrophobic interactions may or redox reactions etc. [8]. This in retrospect may not have have an important role to play. Not only during binding, been such a wise move as we believed till now. even during catalytic steps, the contributions of the ac- In 1955–56, International Commission of enzymes was tive site residues may differ qualitatively and/or quanti- set up and that is how the EC classification came into be- tatively. This “accidental promiscuity” observed in wild ing [8]. As we know, this classification provides a number forms of the enzymes should be distinguished from “in- with four figures. It is worth noting that all the four figures duced promiscuity” exhibited by mutants obtained by relate to the details of the specificity of the enzyme. The protein engineering or [12]. Many ex- firm belief was that if one is able to pin down complete cellent reviews (other than those already quoted above) details of the specificity, one has described the enzyme which include evolutionary aspects of promiscuity are and that is its identity. available [3,13,14].

Various kinds of enzyme promiscuity Condition promiscuity of enzymes Nothing promotes a scientific direction asmuch as coining hydrolyse substrates. What happens if water a new term. System biology, nanotechnology and synthetic is nearly absent? Advent of non-aqueous enzymology biology are illustrative examples. Enzyme promiscuity is showed that exploring this possibility led to carrying out also a case in point. As Hult and Burglund [9] pointed biocatalysis in nearly anhydrous organic solvents [15,16], out, promiscuous catalysis by of reverse micelles [17,18], ionic liquids [19-22] etc. So, a formation of C-C bonds was reported in 1921 and forms in low water containing organic solvent synthe- the basis of a current industrial process. Khersonsky and sizes an ester bond and is obviously not functioning as a Tawfik [4] have also cited examples of few other well [23]. known enzymes which, many decades back, were reported This has been a very useful discovery. It has been also to catalyse reactions “in addition to the ones for which very important as it opened up huge possibilities of they are physiologically specialized or evolve…”. As Babtie using enzymes in biotransformations [16,24] [Figure 1]. et al. [10] highlighted “Promiscuous activities are generally Not only it makes it possible to use inexpensive hydrolases considerably less efficient than the primary function of an like and lipases in organic synthesis, it provides enzyme, but second order rate constants (kcat/KM)ashigh an additional handle of medium engineering [25,26]. Chan- 5 −1 −1 as 10 M s and rate accelerations ((kcat/KM)/k2)upto ging the organic solvent may change the enantioselectivity 1018 have been reported: these values match or exceed [27,28]. The other possibilities emerged e.g. using organic those for many native enzymatic reactions.”. After initial solvents as co-solvents [29] or using biphasic systems con- loose usages of the terms (associated with promiscuous sisting of aqueous medium and water immiscible organic behaviour of enzymes), there is clarity that broad specifi- solvents [30]. One can use organic solvent phase to dis- city of an enzyme in terms of catalysis of the same reac- solve substrates and aqueous phase can contain the en- tion with range of substrates should be called substrate zyme. This makes the catalytic process very efficient in promiscuity. Instances when an enzyme catalyses a differ- case the substrate inhibition is involved [31]. If the prod- ent reaction (which is not believed to be physiologically uct also goes back to the organic phase, shifting of the relevant at that point in evolution) with a different transi- equilibrium (in favour of product formation) and over- tion state should be termed as examples of catalytic prom- coming product inhibition (if involved) are both achieved. iscuity [9,10]. More new possibilities continue to emerge. As Dordick’s Hult and Berglund [9] go a step further and suggest that group showed few years back, nanotube mediated assem- reverse reactions catalysed by enzymes in many non- bly of enzymes at the interface of aqueous and organic aqueous media or co-solvents can be classified as condition media overcomes the transport limitations typical of such promiscuity. In such cases, the transition state may be same biphasic systems [32]. as in normal reactions. So, this may be little confusing. It incidentally also meant that one could use green sol- Enzyme promiscuity started as being perceived as “asso- vents for biocatalysis such as glycerol, ethanol, succinate ciated with unwanted side effects, poor catalytic properties esters, lactate esters, limonene and supercritical fluids anderrorsinbiologicalfunction” [11]. Today, it is increas- [34]. The case of glycerol is especially relevant in the con- ingly being perceived as immensely useful phenomenon text of white biotechnology as massive amounts of gly- which can dramatically enhance utility of biocatalysis in cerol are generated during biodiesel production and hence biotechnology. fits in well with the biorefinery concept. The biodiesel as Babtie et al. [10] have provided a good discussion on such can be obtained from wastes and can be a good ex- how catalytic promiscuity may operate. Apart from the ample of waste valorization [35-37] [Figure 2]. Many of different amino acids of the involved in a the above mentioned solvents also, in principle, can be ob- qualitative or quantitative fashion during binding of the tained from non food biomass or waste food materials Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 3 of 9

Figure 1 Lipase catalyzed transesterification of α,β-glucose or α,β-maltose with vinyl propionate or ethyl acrylate as acyl donor produced monoesters and diesters. α-Cyclodextrin and the mentioned ester products, were substrates in the second reaction catalyzed by CGTase from B. macerans to produce oligosaccharide esters. [Reproduced from Ref [33] with permission from Chemistry Central].

[35]. All this points out to the emerging contours of a review is on illustrating how catalytic promiscuity can be chemical industry based upon sustainable practices. exploited to create novel possibilities in the area of bioca- These possibilities have emerged as our classical con- talysis driving the growth of white biotechnology. Some ex- cept of hydrolases will be always hydrolases turned out amples of reactions carried out using catalytic promiscuity to be not absolutely valid. If you change the reaction for the synthesis of organic compounds in the authors’ la- condition, enzymes show condition promiscuity. That boratory are illustrated in Table 1. Also, we have mostly has not turned out to be bad at all from the perspective limited ourselves to examples of accidental promiscuity ra- of white biotechnology. ther than including ever growing number of examples where catalytic promiscuity has been tailored by using pro- Catalytic promiscuity: recent results tein engineering or directed evolution. As many examples of catalytic promiscuity have been cov- ered in quite a few recent reviews [5,38,39], we will mostly Catalytic promiscuity of lipases focus on literature pertaining to last couple of years. Rather Single pot multicomponent reactions are an efficient syn- than aiming at being comprehensive, the thrust of this thetic strategy especially if accompanied by good atom

Figure 2 Biodiesel conversion with clean oil using Novozym 435 + RMIM. Reactions were performed taking 0.5 g coffee oil. Ethanol was added in molar ratio 4:1 (ethanol: oil). Novozym 435 + RMIM = 3:1 (White Bars); 37.5 mg of enzyme Novozym 435 (enzyme load being 7.5% w/w of oil) and 12.5 mg of enzyme RMIM (enzyme load being 2.5% w/w of oil) was added. Novozym 435 + RMIM = 1:1(Grey Bars); 25 mg of enzyme Novozym 435 (enzyme load being 5% w/w of oil) and 25 mg of enzyme RMIM (enzyme load being 5% w/w of oil) was added. Novozym 435 + RMIM = 1:3 (Black bars); 12.5 mg of enzyme Novozym 435 (enzyme load being 2.5% w/w of oil) and 37.5 mg of enzyme RMIM (enzyme load being 7.5% w/w of oil) was added. [Reproduced from Ref [37] with permission from Chemistry Central]. Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 4 of 9

Table 1 Some interesting examples of organic compounds synthesized in the authors’ laboratory exploiting catalytic promiscuity Structure of the compound Substrate and reaction conditions Reference Substrate: 5-Hydroxy-endo tricyclo [5.2.1.02,6] deca-4,8-dien-3-one and vinyl acetate [40] Biocatalyst: Novozyme 435 Solvent: Vinyl acetate (substrate) containing 10 % (v/v) pyridine/DMF

Substrate: p-nitrobenzaldehyde and ethyl acetoacetate [41] Biocatalyst: Lipases from Candida antarctica lipase B, Mucor javanicus, Mucor miehei, Candida rugosa and Novozyme 435 Solvent: Aqueous organic co-solvent mixtures were used as the solvent

Substrate: p-nitrobenzaldehyde and acetyl acetone [42] Biocatalyst: Lipases from Candida antarctica lipase B, Mucor javanicus, Mucor miehei, Burkholderia cepacia and Novozyme 435 Solvent: Aqueous organic co-solvent mixtures

Substrate: p-nitrobenzaldehyde [43] Biocatalyst: Novozyme 435 Solvent: Aqueous buffer Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 5 of 9

economy. Zhang [44] showed that porcine pancreatic under solvent-free conditions. The catalytic promiscuity lipase (PPL) could be used to synthesize a number of of Escherichia coli BioH was recently exploited 2, 4-disubstituted thiazoles with methanol as the organic for the synthesis of 3, 4-dihydropyran derivatives [52]. The solvent. Their interest in synthesis of these compounds was authors used a series of substituted benzalacetones and 1, due to their many biological activities. There are several 3-cyclic diketones as reactants in anhydrous DMF, with noteworthy features of this work. Use of organic solvents yields as high as 76% being obtained in some cases. Re- (as compared to water or aqueous-organic co-solvent mix- cently, ficin from fig tree latex (a plant cysteine proteinase) tures) has been underexploited in the context of catalytic was shown to catalyze the direct asymmetric aldol reac- promiscuity of enzymes. More important, some tions of nitrogen, oxygen or sulphur containing heterocyc- preparations have also been shown to be catalysing this re- lic ketones with aromatic aldehydes in organic medium action. It is very likely that enzymes used in this work [53]. Earlier work from the same group showed that com- were industrial grade preparations and may have been mercially available (from Carica papaya latex) can contaminated by lipase activities. Earlier, it took several be used as an efficient catalyst for Knoevenagel reactions decades before Nakajima’s group could show that lipases involving a wide range of substrates [54]. Zheng et al. [55] (contrary to an earlier claim) if purified do not catalyse developed the trypsin catalyzed one-pot multicomponent peptide synthesis in organic solvents [45]. As we explore synthesis of 4-thiazolidinones as a novel strategy for the more and more catalytic promiscuity of enzymes, it is ne- synthesis of this important group of heterocyclic com- cessary that we keep in mind that some of these may be pounds. The derivatives of 2H-1-benzopyran-2-one form caused by contaminating proteins. Another important the scaffold of many pharmaceuticals [56,57]. Wang et al. point is that methanol as a solvent was far superior to [58] have used an alkaline from B. licheniformis ethanol. That is unfortunate as ethanol, unlike methanol, to obtain these by domino Knoevanagel/intramolecular is a green solvent. So, from sustainability point of view, transesterification reactions in low water containing or- perhaps we need to separately screen out a panel of green ganic solvents. solvents and determine which one is the best, even if not An interesting example of exploiting catalytic promiscu- as good as another non green solvent. ity for carrying out domino, single-pot reactions was re- Ugi reaction is one of the more well known multicom- ported by Zhou et al. [59]. The authors used α-amylase ponent reactions [46]. The classic Ugi reaction involves from Bacillus subtilis for catalyzing the oxa-Michael/aldol condensation of a primary amine, a carbonyl compound, condensation for the synthesis of substituted chromene carboxylic acid and isocyanide. Kzossowski et al. [47] have derivatives. Gao et al. [60] described Henry reactions cata- shown that Novozym 435 could form a peptide bond lyzed by glucoamylase from Aspergillus niger. The reac- in organic solvents like toluene and chloroform. An tions carried out in mixed solvents of ethanol and water, important objective of white biotechnology has been formed the β-nitroalcohols in yields upto 99%. to be able to operate chemical processes starting with One emerging concern has been the bacteria develop- simple set of compounds [34] which can be obtained ing resistance towards existing antibiotics like in the case from renewable resources. In that respect, peptide bond of aminoglycosides which are broad spectrum antibiotics formation starting with an amine, aldehyde and iso- [61]. The catalytic promiscuity of aminoglycoside acetyl cyanide (rather than two amino acids) is a good advance- transesterases was utilized for chemoenzymatic synthesis ment and shows the wide ranging promise and potential of variety of novel molecules including acylated amino- of catalytic promiscuity. glucosides which show promise in circumventing bacterial resistance towards existing antibiotics. The synthetic strat- Catalytic promiscuity: beyond lipases egy avoids longer multistep processes. Werneburg et al. The phenomenon of catalytic promiscuity has turned out [62] have used the polyketide synthetase for preparative to be far more widespread than initially believed. While scale synthesis of 15 new aureothin (a shikimate-polyketide earlier, there was a strong focus on the use of lipases for with antimicrobial and antitumor activities) analogs, many C-C bond formation reactions [38,40,41,48,49], use of with less cytotoxicity but improved anti-proliferative other enzymes in catalyzing diverse kinds of reactions is action. It may be noted that engineered mutants of the being increasingly demonstrated. Liu et al. [50] have re- organism were used. So, it is an example of metabolic en- ported asymmetric aldol reactions between isatin deriva- gineering wherein the strategy was based upon the cata- tives with cyclic ketones catalyzed by p1 from lytic promiscuity of the enzyme. Penicillium citrinum. This example of catalytic promis- Alcohol (ADHs) are enzymes which cuity provides a green approach for the synthesis of pharma- catalyze the transformation of ketones/aldehydes to the ceutically active compounds. Li et al. [51] had earlier used corresponding alcohols and vice versa at the expense the same enzyme for catalyzing asymmetric aldol reac- of a nicotinamide that acts as hydride donor tions between aromatic aldehydes and cyclic ketones and acceptor respectively [63]. Of the many approaches Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 6 of 9

available for co-factor recycling, a simple way is to use a Similarly, Baas et al. [75] have reviewed the enzyme co-substrate such as propanol [64,65]. Gotor’sgroup[66] promiscuity in five members of the tautomerase super has used ADHs from L. brevis, R.ruber and Thermoanaer- family. These enzymes show diverse catalytic activities obacter sp. to carry out regioselective and stereoselective encompassing C-H, C-C, C-O and carbon-halogen bonds. reductions of 1, 2-and 1,3- diketones to obtain enantio- The review of Baas et al. [75] discusses how looking at pure hydroxyketones or diols. Some cyclic diketones were promiscuous activities helps in understanding of mechan- also reduced. The co-substrate propanol was used for co- ism of normal activities of the enzymes. It is interesting factor regeneration in this case as well. While the reaction that all enzymes have N-terminal proline as a key catalytic carried out was essentially a normal one, the binding residue. It is very well established that some amino acids modes of the substrates were different with different as such catalyse diverse kinds of reactions on their own ADHs and show how different parts of the active site of [76,77].Obviously, the catalytic promiscuity of enzymes the enzymes can be involved in binding. This is a good il- has its origin in confluence of different factors [11]. Table 2 lustration of substrate promiscuity being exploited for de- summarizes the usefulness of catalytic promiscuity in bio- signing useful synthetic strategies. An interesting example catalysis (Table 2). of catalytic chemo-promiscuity of alcohol Archae constitute the oldest organisms on our earth. was reported by Ferreira-Silve et al. [67]. The authors ob- Given the harsher conditions prevalent in those days, served that some alcohol dehydrogenases transformed archae are prominent examples of extremophiles. The phenylacetaldoxime to the primary alcohol via the imine specialised functions of the enzymes (part of the evolved and aldehyde intermediates, suggesting that the hydride of metabolism) in more complex organism have evolved the co-factor was transferred to the N-atom of the oxime from the small number of enzymes which these ances- moiety rather than the C-atom. tors had. So, these organisms constitute valuable systems is a key PLP-dependant enzyme in to track extensive promiscuity shown by the early en- cyclosporine (a well known immunosuppressant drug) zymes. Jia et al. have detailed both promiscuity as well as biosynthesis. Di Salvo et al. [68] reported that just like moonlighting shown by archae enzymes [82]. Archae en- serine hydroxymethyl and , zymes are rich in intrinsic disorder. Jia et al. [82] point the cloned racemase was able to carry out both retroal- out that capacity for the structure to survive under harsh dol cleavage and transamination reactions. It is a strong conditions and multitasking (which is seen as promiscu- evidence that these three enzymes illustrate divergent ity and moonlighting) required conformational pliability. evolution from a common ancestor which perhaps singly It is interesting that many “hub proteins” important in performed all these individual reactions. So, the specia- metabolic regulations in many organisms have been found lised functions evolved but the enzymes retained features to be intrinsically disordered proteins (IDPs) [83]. So, which are responsible for the promiscuous behaviour. promiscuity is one facet of the evolutionary design of en- A novel protocol for the D- catalyzed zymes. As Skolnick et al. [84] state, promiscuous behaviour double Michael addition was developed by Chen et al. is the biochemical noise (low level, ligand-protein interac- [69]. The reactions, which were used for the synthesis of tions) which was nearly impossible to eliminate as enzyme (hetero)spiro[5.5] undecane derivatives produced the cis molecules evolved to assume specialised catalytic roles. isomers in all the cases. Grulich et al. [70] provide a The example of glutathione transferase is especially in- useful summary of the applications of Penicillin G acy- teresting. Atkins group [85] have discussed that two iso- lases which are known to catalyse transesterifications, forms of glutathione transfereases in humans vastly Markonikov additions or Henry reactions. Unfortunately, differ in their catalytic promiscuity. This highlights the the catalytic efficiency reported so far in these promiscu- conceptual relationship between the phenomenon of iso- ous reactions is far from satisfactory for any biotechno- enzymes to promiscuous behaviour as pointed out by one logical applications. However, as Nobeli et al. [11] had of us few years back [5]. The A-class GSTA1-1 showed the pointed out, such results lay the foundation of subsequent fairly wide substrate specificity as a detoxification enzyme. efforts using protein engineering/ directed evolution to On the other hand, GSTA4-4 acted upon lipid peroxida- improve upon these catalytic activities. Large number of tion products. This was one of the early reports to point successful results which prove this are already available in out that “conformational plasticity” is inbuilt to achieve the literature [12,71-73]. catalytic promiscuity at the cost of stability [85]. More He et al. [74] reported for the first time that hen egg recent work from the same group [86] concludes that white lysozyme (HEWL) efficiently promotes the one- smooth barrier free transitions within the local conform- pot, three-component aza-Diels-Alder reaction of aro- ational landscape of the active site is associated with the matic aldehydes, aromatic amine and 2-cyclohexen-1-one. more promiscuous GST. Furthermore, local molten glob- Under optimised conditions, yields up to 98% and stereo- ule behaviour optimizes the catalytic function of the GST selectivity of endo/exo ratios up to 90:10 were obtained. in detoxification [87]. So, catalytic promiscuity may not be Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 7 of 9

Table 2 Applications of catalytic promiscuity for useful biotransformations Biotransformations Substrate (s) Enzyme (s) References Regio- and stereoselective Diketones ADHs from Lactobacillus kefir, Rhodococcus [66] reductions ruber, Thermoanaerobacter etc. Reduction reaction Phenylacetaldoxime ADHs from Rhodococcus ruber, Ralstonia sp., [67] Thermoanaerobacter etc. Aza-Diels-Alder reaction 4-chlorobenzaldehyde, cyclohexenone, 4-anisidine Hen egg white lysozyme (HEWL) [74] Domino Knoevenagel/ Salicylaldehyde, ethyl acetoacetate Alkaline protease from Bacillus [58] intramolecular transesterification licheniformis (BLAP) Transesterification reaction Guaifenesin, vinyl acetate Penicillin G acylase [78] Knoevenagel reaction Aromatic aldehyde, acetyl acetone/Ethyl acetoacetate Papain from Carica papaya latex [54] Ugi reaction for peptide Aldehyde, amine, isocyanide Novozyme 435 (commercially available, [47] synthesis (MCR)* immobilized Candida antarctica lipase B) Asymmetric aldol reaction Aromatic aldehyde, cyclic ketone Nuclease p1 from Penicillium citrinum [51] Asymmetric aldol reaction Heterocyclic ketone, aromatic aldehyde Ficin from fig tree latex [53] Synthesis of 4-thiazolidinones Aromatic aldehyde, benzyl amine, mercaptoacetic acid Trypsin from porcine pancreas [55] Domino oxa-Michael/aldol Salicylaldehyde, methyl vinyl ketone α-amylase from Bacillus subtilis [59] condensations Henry reaction 4-cyanobenzaldehyde, nitromethane Glucoamylase from Aspergillus niger (AnGA) [60] Retro aldol and transamination L-threonine and L-allo-threonine (for retro aldol Alanine racemase from Tolypocladium [68] reactions reaction); D- and L-alanine(for transamination reaction) inflatum Double Michael addition reaction Cyclohexane-1,3-dione and (1E,4E)-1, D-amino acylase [69] 5-diphenylpenta-1,4-dien-3-one Enantioselective aldol reaction Isatin derivatives, cyclic ketones Nuclease p1 from Penicillium citrinum [50] Synthesis of 2,4-disubstituted Benzylamine, isobutyraldehyde, thioacetic acid, Porcine pancreatic lipase (PPL) [44] thiazoles (MCR)* methyl 3-(dimethylamino)-2-isocyanoacrylate Michael addition-cyclization cas- Substituted benzalacetones and 1,3-cyclic diketones E.coli BioH esterase [52] cade reaction Synthesis of substituted Salicylaldehyde, acetophenone, methanol Porcine pancreatic lipase [79] 2H-chromemes (MCR)* Baylis-Hillman reaction p-nitrobenzaldehyde and methyl vinyl ketone E.coli BioH esterase [80] Biginelli reaction (MCR)* Urea, ethyl acetoacetate, vinyl acetate Trypsin from porcine pancreas [81] *stands for Multi-component reaction. just accidental “noise” [84] but part of an intentional bio- with applications of lipases; last few years have seen catalytic design. other classes of enzymes (other hydrolases, oxidoreduc- Labrou’s group [88] have recently reported a GST from tases, etc.) being equally capable of showing A. tumefaciens which represents a novel class of bacter- catalytic promiscuity. ial GST superfamily. Its active site is quite different from The shift from our belief in enzyme specificity to the cytosolic GST reported so far. It may be interesting to realization that these biocatalysts are fairly promiscuous see the unravelling of its specificity in the years to come. has not been gradual. Some key milestones can be identi- Finally, as pointed out by them, GSTs are also possibly fied. We accepted the idea of broad specificity (lately called involved in the storage and transportation of wide variety relaxed specificity) long ago. Isoforms or isoenzymes, the of biological molecules and thus are also moonlighting enzymes from the same organism, carrying similar catalytic proteins. This three way correlation between isoenzymes, activity but with different specificity and kinetic behaviour promiscuous enzymes and moonlighting proteins have have been again known since several decades [5]. The ca- been pointed out earlier [5]. To sum up, the conform- talysis starts with molecular recognition of the “substrate” ational pliability may be local or global in protein struc- by the enzyme. The , part of the active centre ture as the cause behind promiscuity. was known to show promiscuous behaviour when tex- tile dyes emerged as powerful affinity ligands [89]. The Conclusions catalytic promiscuity largely arises because many dif- Few trends are clear. While during early few years, ex- ferent “substrates” can interact with different side chains amples of catalytic promiscuity were mostly concerned of amino acids to result in different transition states. Arora et al. Sustainable Chemical Processes (2014) 2:25 Page 8 of 9

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